Silva Sütt

Wfs1-deficient mice display impaired behavioural adaptation in stressful environment

Wfs1-deficient mice were generated by disrupting the 8th exon of Wfs1 gene. Reproduction rates of homozygous Wfs1-deficient mice were slightly below the expected values, they displayed intolerance to glucose and overall lower body weight. The present behavioural study was performed in female Wfs1-deficient mice due to their milder metabolic disturbances. Non-fasting blood glucose levels did not differ between homozygous Wfs1-deficient mice and wild-type littermates. While there was no difference in baseline plasma corticosterone, exposure to stress induced a nearly three-fold elevation of corticosterone in Wfs1-deficient mice in relation to wild-type littermates. Wfs1-deficient mice did not display obvious shortcomings in sensory and motor functioning as exemplified by intact responses in conditioned learning paradigms and rota-rod test. Locomotor activity of Wfs1-deficient mice was significantly lower only in brightly lit environment. Short-term isolation had a significant anxiogenic-like effect on the behaviour of Wfs1-deficient mice in dark/light exploration test. Lower exploratory activity of Wfs1-deficient mice in the plus-maze was antagonised by pre-treatment with diazepam (1 mg/kg), a GABA(A) receptor agonist. Wfs1-deficient mice displayed increased anxiety-like behaviour in hyponeophagia test. The locomotor stimulatory effects of amphetamine (2.5-7.5 mg/kg) and apomorphine (3 mg/kg) were significantly attenuated and facilitated, respectively, in Wfs1-deficient mice. There were no differences between Wfs1-deficient mice and wild-types in forced swimming behaviour and conditioned fear responses. Subtle impairments in reversal learning were apparent in Wfs1-deficient mice in the Morris water maze. Altogether, the present study demonstrates impaired behavioural adaptation of Wfs1-deficient mice in stress-inducing situations. It is likely that Wfs1 protein plays a major role in the behavioural adaptation mechanisms to novel and stressful environments.

Relation between increased anxiety and reduced expression of alpha1 and alpha2 subunits of GABAA receptors in Wfs1-deficient mice

Mutations in the coding region of the WFS1 gene cause Wolfram syndrome, a rare multisystem neurodegenerative disorder of autosomal recessive inheritance. In clinical studies a relation between mutations in the Wfs1 gene and increased susceptibility for mood disorders has been established. According to our previous studies, mice lacking Wfs1 gene displayed increased anxiety in stressful environment. As the GABA-ergic system plays a significant role in the regulation of anxiety, we analyzed the expression of GABA-related genes in the forebrain structures of wild-type and Wfs1-deficient mice. Experimentally naïve Wfs1-deficient animals displayed a significant down-regulation of alpha1 (Gabra1) and alpha2 (Gabra2) subunits of GABA(A) receptors in the temporal lobe and frontal cortex. Exposure of wild-type mice to the elevated plus-maze decreased levels of Gabra1 and Gabra2 genes in the temporal lobe. A similar tendency was also established in the frontal cortex of wild-type animals exposed to behavioral test. In Wfs1-deficient mice the elevated plus-maze exposure did not induce further changes in the expression of Gabra1 and Gabra2 genes. By contrast, the expression of Gad1 and Gad2 genes, enzymes responsible for the synthesis of GABA, was not significantly affected by the exposure of mice to the elevated plus-maze or by the invalidation of Wfs1 gene. Altogether, the present study demonstrates that increased anxiety of Wfs1-deficient mice is probably linked to reduced expression of Gabra1 and Gabra2 genes in the frontal cortex and temporal lobe.

Lsamp⁻/⁻ mice display lower sensitivity to amphetamine and have elevated 5-HT turnover.

In mice, the limbic system-associated membrane protein (Lsamp) gene has been implicated in locomotion, anxiety, fear reaction, learning, social behaviour and adaptation. Human data links the LSAMP gene to several psychiatric disorders and completed suicide. Here, we investigated changes in major monoamine systems in mice lacking the Lsamp gene. First, the locomotor and rewarding effects of amphetamine were studied in Lsamp(-/-) mice and Lsamp(+/+) mice. Second, monoamine levels in major brain regions in response to saline and amphetamine injections were measured and, third, the expression levels of dopamine system-related genes in the brain were studied in these mice. Lsamp(-/-) mice displayed lower sensitivity to amphetamine in the motility box. Likewise, in the place preference test, the rewarding effect of amphetamine was absent in Lsamp(-/-) mice. In all brain regions studied, Lsamp(-/-) mice displayed lower serotonin (5-HT) baseline levels, but a greater 5-HT turnover rate, and amphetamine increased the level of 5-HT and lowered 5-HT turnover to a greater extent in Lsamp(-/-) mice. Finally, Lsamp(-/-) mice had lower level of dopamine transporter (DAT) mRNA in the mesencephalon. In conclusion, Lsamp-deficiency leads to increased endogenous 5-HT-ergic tone and enhanced 5-HT release in response to amphetamine. Elevated 5-HT function and reduced activity of DAT are the probable reasons for the blunted effects of amphetamine in these mice. Lsamp(-/-) mice are a promising model to study the neurobiological mechanisms of deviant social behaviour and adaptation impairment observed in many psychiatric disorders.

Evidence for impaired function of dopaminergic system in Wfs1-deficient mice.

Immunohistological studies suggest abundant expression of Wfs1 protein in neurons and nerve fibers that lie in the vicinity of dopaminergic (DA-ergic) fibers and neurons. Therefore, we sought to characterize the function of DA-ergic system in Wfs1-deficient mice. In wild-type mice, amphetamine, an indirect agonist of DA, caused significant hyperlocomotion and increase in tissue DA levels in the dorsal and ventral striatum. Both effects of amphetamine were significantly blunted in homozygous Wfs1-deficient mice. Motor stimulation caused by apomorphine, a direct DA receptor agonist, was somewhat stronger in Wfs1-deficient mice compared to their wild-type littermates. However, apomorphine caused a similar reduction in levels of DA metabolites (3,4-dihydroxyphenylacetic acid and homovanillic acid) in the dorsal and ventral striatum in all genotypes. Behavioral sensitization to repeated treatment with amphetamine (2.5 mg/kg) was observed in wild-type, but not in Wfs1-deficient mice. The expression of DA transporter gene (Dat) mRNA was significantly lower in the midbrain of male and female homozygous mice compared to wild-type littermates. Altogether, the blunted effects of amphetamine and the reduced gene expression of DA transporter are probably indicative of an impaired functioning of the DA-ergic system in Wfs1-deficient mice.

Trib3 Is Developmentally and Nutritionally Regulated in the Brain but Is Dispensable for Spatial Memory, Fear Conditioning and Sensing of Amino Acid-Imbalanced Diet

Tribbles homolog 3 (TRIB3) is a mammalian pseudokinase that is induced in neuronal cell cultures in response to cell death-inducing stresses, including neurotrophic factor deprivation. TRIB3 is an inhibitor of activating transcription factor 4 (ATF4), the central transcriptional regulator in the eukaryotic translation initiation factor 2α (eIF2α) phosphorylation pathway that is involved in the cellular stress response and behavioral processes. In this article, we study the expression of Trib3 in the mouse brain, characterize the brain morphology of mice with a genetic ablation of Trib3 and investigate whether Trib3 deficiency alters eIF2α-dependent cognitive abilities. Our data show that the consumption of a leucine-deficient diet induces Trib3 expression in the anterior piriform cortex, the brain region responsible for detecting essential amino acid intake imbalance. However, the aversive response to leucine-devoid diet does not differ in Trib3 knockout and wild type mice. Trib3 deletion also does not affect long-term spatial memory and reversal learning in the Morris water maze and auditory or contextual fear conditioning. During embryonic development, Trib3 expression increases in the brain and persists in the early postnatal stadium. Neuroanatomical characterization of mice lacking Trib3 revealed enlarged lateral ventricles. Thus, although the absence of Trib3 does not alter the eIF2α pathway-dependent cognitive functions of several areas of the brain, including the hippocampus, amygdala and anterior piriform cortex, Trib3 may serve a role in other central nervous system processes and molecular pathways.

Wfs1-deficient animals have brain-region-specific changes of Na+, K+-ATPase activity and mRNA expression of α1 and β1 subunits

Mutations in the WFS1 gene, which encodes the endoplasmic reticulum (ER) glycoprotein, cause Wolfram syndrome, a disease characterized by juvenile‐onset diabetes mellitus, optic atrophy, deafness, and different psychiatric abnormalities. Loss of neuronal cells and pancreatic β‐cells in Wolfram syndrome patients is probably related to the dysfunction of ER stress regulation, which leads to cell apoptosis. The present study shows that Wfs1‐deficient mice have brain‐region‐specific changes in Na+,K+‐ATPase activity and in the expression of the α1 and β1 subunits. We found a significant (1.6‐fold) increase of Na‐pump activity and β1 subunit mRNA expression in mice lacking the Wfs1 gene in the temporal lobe compared with their wild‐type littermates. By contrast, exposure of mice to the elevated plus maze (EPM) model of anxiety decreased Na‐pump activity 1.3‐fold in the midbrain and dorsal striatum and 2.0‐fold in the ventral striatum of homozygous animals compared with the nonexposed group. Na‐pump α1‐subunit mRNA was significantly decreased in the dorsal striatum and midbrain of Wfs1‐deficient homozygous animals compared with wild‐type littermates. In the temporal lobe, an increase in the activity of the Na‐pump is probably related to increased anxiety established in Wfs1‐deficient mice, whereas the blunted dopamine function in the forebrain of Wfs1‐deficient mice may be associated with a decrease of Na‐pump activity in the dorsal and ventral striatum and in the midbrain after exposure to the EPM.

Effect of chronic valproic acid treatment on hepatic gene expression profile in wfs1 knockout mouse

Valproic acid (VPA) is a widely used anticonvulsant and mood-stabilizing drug whose use is often associated with drug-induced weight gain. Treatment with VPA has been shown to upregulate Wfs1 expression in vitro. Aim of the present study was to compare the effect of chronic VPA treatment in wild type (WT) and Wfs1 knockout (KO) mice on hepatic gene expression profile. Wild type, Wfs1 heterozygous, and homozygous mice were treated with VPA for three months (300 mg/kg i.p. daily) and gene expression profiles in liver were evaluated using Affymetrix Mouse GeneChip 1.0 ST array. We identified 42 genes affected by Wfs1 genotype, 10 genes regulated by VPA treatment, and 9 genes whose regulation by VPA was dependent on genotype. Among the genes that were regulated differentially by VPA depending on genotype was peroxisome proliferator-activated receptor delta (Ppard), whose expression was upregulated in response to VPA treatment in WT, but not in Wfs1 KO mice. Thus, regulation of Ppard by VPA is dependent on Wfs1 genotype.

Wfs1-deficient mice display altered function of serotonergic system and increased behavioral response to antidepressants

It has been shown that mutations in the WFS1 gene make humans more susceptible to mood disorders. Besides that, mood disorders are associated with alterations in the activity of serotonergic and noradrenergic systems. Therefore, in this study, the effects of imipramine, an inhibitor of serotonin (5-HT) and noradrenaline (NA) reuptake, and paroxetine, a selective inhibitor of 5-HT reuptake, were studied in tests of behavioral despair. The tail suspension test (TST) and forced swimming test (FST) were performed in Wfs1-deficient mice. Simultaneously, gene expression and monoamine metabolism studies were conducted to evaluate changes in 5-HT- and NA-ergic systems of Wfs1-deficient mice. The basal immobility time of Wfs1-deficient mice in TST and FST did not differ from that of their wild-type littermates. However, a significant reduction of immobility time in response to lower doses of imipramine and paroxetine was observed in homozygous Wfs1-deficient mice, but not in their wild-type littermates. In gene expression studies, the levels of 5-HT transporter (SERT) were significantly reduced in the pons of homozygous animals. Monoamine metabolism was assayed separately in the dorsal and ventral striatum of naive mice and mice exposed for 30 min to brightly lit motility boxes. We found that this aversive challenge caused a significant increase in the levels of 5-HT and 5-hydroxyindoleacetic acid (5-HIAA), a metabolite of 5-HT, in the ventral and dorsal striatum of wild-type mice, but not in their homozygous littermates. Taken together, the blunted 5-HT metabolism and reduced levels of SERT are a likely reason for the elevated sensitivity of these mice to the action of imipramine and paroxetine. These changes in the pharmacological and neurochemical phenotype of Wfs1-deficient mice may help to explain the increased susceptibility of Wolfram syndrome patients to depressive states.

Relation of exploratory behaviour to plasma corticosterone and Wfs1 gene expression in Wistar rats

Male Wistar rats exhibit significant variations in exploratory behaviour in the elevated plus-maze (EPM) model of anxiety. We have now investigated the relation between exploratory behaviour and levels of corticosterone and systemic oxidative stress. Also, the expression levels of endocannabinoid-related and wolframin (Wfs1) genes were measured in the forebrain structures. The rats were divided into high, intermediate and low exploratory activity groups. Exposure to EPM significantly elevated the serum levels of corticosterone in all rats, but especially in the high exploratory group. Oxidative stress indices and expression of endocannabinoid-related genes were not significantly affected by exposure to EPM. Wfs1 mRNA level was highly dependent on exploratory behaviour of animals. In low exploratory activity rats, Wfs1 gene expression was reduced in the temporal lobe, whereas in high exploratory activity group it was reduced in the mesolimbic area and hippocampus. Altogether, present study indicates that in high exploratory activity rats, the activation of brain areas related to novelty seeking is apparent, whereas in low exploratory activity group the brain structures linked to anxiety are activated.

Cat odor exposure induces distinct changes in the exploratory behavior and Wfs1 gene expression in C57Bl/6 and 129Sv mice

129Sv and C57Bl/6 (Bl6) strains are two most widely used inbred mice strains for generation of transgenic animals. The present study confirms the existence of substantial differences in the behavior of these two mice strains. The exploratory behavior of Bl6 mice in a novel environment was significantly higher compared to 129Sv mice. The exposure of mice to cat odor-induced an anxiety-like state in Bl6, but not in 129Sv mice. The levels of Wfs1 gene expression did not differ in the prefrontal cortex, mesolimbic area and temporal lobe of experimentally naive Bl6 and 129Sv mice. However, after cat odor exposure the expression of Wfs1 gene was significantly lower in the mesolimbic area and temporal lobe of Bl6 mice compared to 129Sv strain. Dynamics of Wfs1 gene expression and exploratory behavior suggest that the down-regulation of Wfs1 gene in Bl6 mice might be related to the increased anxiety. Further studies are needed to test the robustness and possible causal relationship of this finding.